97 results on '"Wickremasinghe RG"'
Search Results
2. Retinoic acid-induced granulocytic differentiation of HL60 human promyelocytic leukemia cells is preceded by downregulation of autonomous generation of inositol lipid-derived second messengers
- Author
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Porfiri, E, primary, Hoffbrand, AV, additional, and Wickremasinghe, RG, additional
- Published
- 1991
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3. Binding of G-CSF, GM-CSF, tumor necrosis factor-alpha, and gamma- interferon to cell surface receptors on human myeloid leukemia cells triggers rapid tyrosine and serine phosphorylation of a 75-Kd protein
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Evans, JP, primary, Mire-Sluis, AR, additional, Hoffbrand, AV, additional, and Wickremasinghe, RG, additional
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- 1990
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4. Editorial: Phospholipases C and A2 in malignant cell proliferation
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Wickremasinghe Rg and Jeremy Jy
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chemistry.chemical_classification ,Cell division ,Phospholipase C ,Cell growth ,Clinical Biochemistry ,hemic and immune systems ,Cell Biology ,respiratory system ,Biology ,Phospholipase ,Molecular biology ,Cell biology ,Enzyme ,Phospholipase A2 ,GTP-binding protein regulators ,chemistry ,cardiovascular system ,biology.protein ,lipids (amino acids, peptides, and proteins) ,tissues ,Gene - Abstract
Phospholipase C et proteine G. Oncogenes ras et Phospholipase C. Role des eicosanoide dans la proliferation cellulaire
- Published
- 1989
5. Synergistic action of calcium ionophore A23187 and phorbol ester TPA on B-chronic lymphocytic leukemia cells
- Author
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Drexler, HG, Brenner, MK, Coustan-Smith, E, Wickremasinghe, RG, and Hoffbrand, AV
- Abstract
The peripheral blood mononuclear cells from 16 patients with B-chronic lymphocytic leukemia (B-CLL, n = 13), B-prolymphocytic leukemia (B-PLL, n = 2) or hairy cell leukemia (n = 1) were incubated in the presence of the phorbol ester 12–0-tetradecanoylphorbol-13-acetate (TPA) and the calcium ionophore A23187. A synergy between these inducers was found with respect to morphological changes and B cell proliferation and differentiation. A23187 used alone did not activate the cells. B-CLL cells treated with the double stimulus acquired a plasmacytoid morphology, showed significantly higher incorporation of 3H-thymidine and 3H-uridine, and produced significantly higher amounts of monoclonal immunoglobulin compared with the same cells exposed to either of the inducers alone. These results indicate that phorbol ester and calcium ionophore act synergistically on B-CLL cells to induce proliferation and differentiation. B-PLL cells responded more vigorously to the signals provided by TPA and A23187. Previous studies showed that TPA and A23187 can mimic the two physiological second messengers diacylglycerol and inositol trisphosphate in the transduction of signals leading to cell activation, proliferation, and differentiation in normal B cells. The present findings suggest that the capacity of B- CLL and B-PLL cells to differentiate in response to signals of the second messenger pathway is intact.
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- 1987
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6. The effect of folate analogues and vitamin B12 on provision of thymine nucleotides for DNA synthesis in megaloblastic anemia
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Taheri, MR, Wickremasinghe, RG, Jackson, BF, and Hoffbrand, AV
- Abstract
The role of vitamin B12 in the folate dependent biosynthesis of thymidine nucleotides is controversial. In an attempt to clarify this, three methods have been used to assess the relative efficacy of vitamin B12 (hydroxocobalamin) and various folate analogues in titrated concentrations at correcting ‘de novo’ thymidylate synthesis by megaloblastic human marrow cells: (1) The deoxyuridine (dU) suppression test which analyses the reduction in (3H)-thymidine labeling of DNA by unlabeled dU. Marrow cells were also labeled with (6–3H)-dU with assessment of (2) its incorporation into DNA and (3) the accumulation of (6–3H)-deoxyuridine monophosphate (3H-dUMP). The three methods gave similar results. In both, N6-formyl tetrahydrofolate (formyl-FH4) was the most effective agent at correcting thymidylate synthesis in megaloblastic anemia due to vitamin B12 or folate deficiency. Vitamin B12 corrected the lesion in vitamin B12 deficiency but not in folate deficiency. Tetrahydrofolate (FH4) and folic acid were effective in deficiency of vitamin B12 or folate, although in both deficiencies they were less effective than formyl-FH4. Methyl-FH4 was effective in folate deficiency but not in vitamin B12 deficiency. These results confirm the failure of methyl-FH4 utilisation in vitamin B12 deficiency. They suggest that if vitamin B12 is needed in the formylation of FH4, this is a minor role in provision of the correct coenzyme for thymidylate synthesis compared with its major role of provision of FH4 from methyl- FH4.
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- 1982
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7. p53 and Notch signaling in chronic lymphocytic leukemia: clues to identifying novel therapeutic strategies.
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Wickremasinghe RG, Prentice AG, and Steele AJ
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- Animals, Humans, Leukemia, Lymphocytic, Chronic, B-Cell genetics, Receptors, Notch genetics, Tumor Suppressor Protein p53 genetics, Antineoplastic Agents therapeutic use, Leukemia, Lymphocytic, Chronic, B-Cell drug therapy, Leukemia, Lymphocytic, Chronic, B-Cell metabolism, Receptors, Notch metabolism, Signal Transduction drug effects, Tumor Suppressor Protein p53 metabolism
- Abstract
The p53 tumor suppressor protein has a key role in the induction of apoptosis of chronic lymphocytic leukemia (CLL) cells. Abnormalities within the p53 pathway identify a subset of patients with a poor prognosis. This review describes recent advances in understanding the mechanisms that regulate p53 levels and the role of p53 in the control of the cell cycle and of apoptosis. The classical model of p53-mediated apoptosis emphasizes the transcriptional activation of proapoptotic genes. In contrast, a novel model emphasizes p53's non-transcriptional actions as the major route of apoptosis induction, whereas its transcriptional arm predominantly upregulates antiapoptotic genes, thus providing a negative feedback mechanism that limits apoptosis. Further studies have identified the Notch pathway as a candidate p53-induced antiapoptotic mechanism. In contrast to the classical model, the novel model predicts that pharmacological inhibition of p53's transcriptional function or of the Notch signaling pathway will augment apoptosis induction by cytotoxic agents. Therapeutic strategies based on the novel model, which we review here for the first time, may significantly augment the antitumor actions of cytotoxic agents in CLL and in other malignancies.
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- 2011
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8. Differential cytopathology and kinetics of measles oncolysis in two primary B-cell malignancies provides mechanistic insights.
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Patel B, Dey A, Ghorani E, Kumar S, Malam Y, Rai L, Steele AJ, Thomson J, Wickremasinghe RG, Zhang Y, Castleton AZ, and Fielding AK
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- Animals, Apoptosis genetics, Apoptosis physiology, Blotting, Western, Cell Line, Tumor, Cells, Cultured, Chlorocebus aethiops, Humans, Measles virus genetics, Polymerase Chain Reaction, Vero Cells, Xenograft Model Antitumor Assays, Leukemia, Lymphocytic, Chronic, B-Cell therapy, Measles virus physiology, Oncolytic Virotherapy methods, Precursor Cell Lymphoblastic Leukemia-Lymphoma therapy
- Abstract
Clinical trials using vaccine measles virus (MV) as anticancer therapy are already underway. We compared the oncolytic potential of MV in two B-cell malignancies; adult acute lymphoblastic leukemia (ALL, an aggressive leukemia) and chronic lymphocytic leukemia (CLL, an indolent leukemia overexpressing Bcl-2) using patient-derived material. In vitro, distinct cytopathological effects were observed between MV-infected primary ALL and CLL cells, with large multinucleated syncytia forming in ALL cultures compared to minimal cell-to-cell fusion in infected CLL cells. Cell viability and immunoblotting studies confirmed rapid cell death in MV-infected ALL cultures and slower MV oncolysis of CLL cells. In cell lines, overexpression of Bcl-2 diminished MV-induced cell death providing a possible mechanism for the slower kinetic of MV oncolysis in CLL. In vivo, intratumoral MV treatment of established subcutaneous ALL xenografts had striking antitumor activity leading to complete resolution of all tumors. The antitumor activity of MV was also evident in disseminated ALL xenograft models. In summary, both ALL and CLL are targets for MV-mediated lysis albeit with different kinetics. The marked sensitivity of both primary ALL cells and ALL xenografts to MV oncolysis highlights the tremendous potential of MV as a novel replicating-virus therapy for adult ALL.
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- 2011
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9. Aberrantly activated anti-apoptotic signalling mechanisms in chronic lymphocytic leukaemia cells: clues to the identification of novel therapeutic targets.
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Wickremasinghe RG, Prentice AG, and Steele AJ
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- Antineoplastic Agents pharmacology, Apoptosis drug effects, Apoptosis physiology, Enzyme Activation physiology, Enzyme Inhibitors pharmacology, Humans, Intracellular Signaling Peptides and Proteins metabolism, Leukemia, Lymphocytic, Chronic, B-Cell drug therapy, Leukemia, Lymphocytic, Chronic, B-Cell pathology, Molecular Targeted Therapy methods, Protein-Tyrosine Kinases antagonists & inhibitors, Syk Kinase, src-Family Kinases metabolism, Leukemia, Lymphocytic, Chronic, B-Cell enzymology, Protein-Tyrosine Kinases metabolism, Signal Transduction physiology
- Abstract
Chronic lymphocytic leukaemia (CLL) is the commonest haematological malignancy in the western world and is incurable by cytotoxic therapy. Considerable research effort has identified the signal transduction pathways in CLL cells that contribute to anti-apoptotic signalling. Some pathways are constitutively activated in CLL cells but upregulated in normal cells only when protein tyrosine kinases (PTKs) are activated by ligands. This review describes which PTKs are aberrantly activated in CLL cells and are potential targets for inhibition. Additional potential targets within pathways downstream of these PTKs include Mek/Erk, mTorc1, protein kinase C, PI-3 kinase/Akt, nuclear factor-κB and cyclin-dependent protein kinase. Numerous studies have identified chemical agents and antibodies that selectively kill CLL cells, irrespective of their genetic resistance to conventional chemotherapeutic agents, and which can overcome cytoprotective microenvironmental signalling. These studies have resulted in identification of novel therapies, some of which are currently undergoing clinical trials. In vitro and animal model studies and clinical trials could determine which inhibitors of which targets are the likely to be most effective and least toxic either singly or in combination., (© 2011 Blackwell Publishing Ltd.)
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- 2011
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10. The JAK3-selective inhibitor PF-956980 reverses the resistance to cytotoxic agents induced by interleukin-4 treatment of chronic lymphocytic leukemia cells: potential for reversal of cytoprotection by the microenvironment.
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Steele AJ, Prentice AG, Cwynarski K, Hoffbrand AV, Hart SM, Lowdell MW, Samuel ER, and Wickremasinghe RG
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- Apoptosis drug effects, Bone Marrow Cells drug effects, Bone Marrow Cells metabolism, Calcium-Calmodulin-Dependent Protein Kinases antagonists & inhibitors, Cytochromes c metabolism, Extracellular Signal-Regulated MAP Kinases metabolism, Flavonoids pharmacology, Gene Expression Regulation, Leukemic drug effects, Humans, Interleukin-4 pharmacology, Janus Kinase 3 metabolism, Myeloid Cell Leukemia Sequence 1 Protein, Phosphorylation drug effects, Proto-Oncogene Proteins c-bcl-2 genetics, STAT6 Transcription Factor metabolism, Stromal Cells drug effects, Stromal Cells metabolism, T-Lymphocytes drug effects, T-Lymphocytes metabolism, Tumor Cells, Cultured, Tumor Suppressor Protein p53 metabolism, bcl-X Protein genetics, Drug Resistance, Neoplasm drug effects, Interleukin-4 therapeutic use, Janus Kinase 3 antagonists & inhibitors, Leukemia, Lymphocytic, Chronic, B-Cell drug therapy, Pyrimidines pharmacology, Pyrroles pharmacology
- Abstract
Extensive evidence suggests that the malignant cells of chronic lymphocytic leukemia (CLL) patients are in close contact with activated T lymphocytes, which secrete a range of cytoprotective cytokines including interleukin-4 (IL-4). IL-4 induced the rapid phosphorylation and activation of the signal transducer and activator of transcription 6 transcription factor in CLL cells in vitro. Longer incubation with IL-4 resulted in up-regulation of the antiapoptotic proteins, Mcl-1 and Bcl-X(L). All of these events were blocked by the JAK3-selective inhibitor, PF-956980. A dye reduction cytotoxicity assay showed that IL-4 induced resistance to the cytotoxic drugs fludarabine and chlorambucil and to the novel p53-elevating agent nutlin 3. IL-4-induced drug resistance was reversed by PF-956980. These conclusions were confirmed by independent assays for apoptosis induction (annexin V binding, cleavage of poly[ADP-ribose] polymerase, and morphologic analysis). Coculture with bone marrow stromal cells in the presence of supernatants derived from activated T-lymphocyte cultures also protected CLL cells from apoptosis induction by chlorambucil. Protection by these combined signals was reversed by PF-956980. The data here provide a preclinical rationale for the possible therapeutic use of PF-956980 in conjunction with conventional cytotoxic drugs to achieve more extensive killing of CLL cells by overcoming antiapoptotic signaling by the microenvironment.
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- 2010
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11. 2-Phenylacetylenesulfonamide (PAS) induces p53-independent apoptotic killing of B-chronic lymphocytic leukemia (CLL) cells.
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Steele AJ, Prentice AG, Hoffbrand AV, Yogashangary BC, Hart SM, Lowdell MW, Samuel ER, North JM, Nacheva EP, Chanalaris A, Kottaridis P, Cwynarski K, and Wickremasinghe RG
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- Aged, Aged, 80 and over, Annexin A5 metabolism, Caspase 3 metabolism, Cytochromes c metabolism, Cytosol metabolism, Cytosol pathology, Dose-Response Relationship, Drug, Drug Resistance drug effects, Drug Screening Assays, Antitumor, Female, Hematopoietic Stem Cells metabolism, Hematopoietic Stem Cells pathology, Humans, Leukemia, Lymphocytic, Chronic, B-Cell metabolism, Leukemia, Lymphocytic, Chronic, B-Cell pathology, Male, Middle Aged, Mitochondria metabolism, Mitochondria pathology, Poly(ADP-ribose) Polymerases metabolism, Protein Transport drug effects, T-Lymphocytes metabolism, T-Lymphocytes pathology, Time Factors, Tumor Cells, Cultured, Up-Regulation drug effects, bcl-2-Associated X Protein metabolism, Antineoplastic Agents pharmacokinetics, Apoptosis drug effects, Gene Expression Regulation, Leukemic drug effects, Leukemia, Lymphocytic, Chronic, B-Cell drug therapy, Proto-Oncogene Proteins c-bcl-2 biosynthesis, Sulfonamides pharmacology, Tumor Suppressor Protein p53 metabolism
- Abstract
We studied the actions of 2-phenylacetylenesulfonamide (PAS) on B-chronic lymphocytic leukemia (CLL) cells. PAS (5-20 microM) initiated apoptosis within 24 hours, with maximal death at 48 hours asassessed by morphology, cleavage of poly(ADP-ribose) polymerase (PARP), caspase 3 activation, and annexin V staining. PAS treatment induced Bax proapoptotic conformational change, Bax movement from the cytosol to the mitochondria, and cytochrome c release, indicating that PAS induced apoptosis via the mitochondrial pathway. PAS induced approximately 3-fold up-regulation of proapoptotic Noxa protein and mRNA levels. In addition, Noxa was found unexpectedly to be bound to Bcl-2 in PAS-treated cells. PAS treatment of CLL cells failed to up-regulate p53, suggesting that PAS induced apoptosis independently of p53. Furthermore, PAS induced apoptosis in CLL isolates with p53 gene deletion in more than 97% of cells. Normal B lymphocytes were as sensitive to PAS-induced Noxa up-regulation and apoptosis as were CLL cells. However, both T lymphocytes and bone marrow hematopoietic progenitor cells were relatively resistant to PAS. Our data suggest that PAS may represent a novel class of drug that induces apoptosis in CLL cells independently of p53 status by a mechanism involving Noxa up-regulation.
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- 2009
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12. Involvement of Tis11b, an AU-rich binding protein, in induction of apoptosis by rituximab in B cell chronic lymphocytic leukemia cells.
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Baou M, Jewell A, Muthurania A, Wickremasinghe RG, Yong KL, Carr R, Marsh P, and Murphy JJ
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- Antibodies, Monoclonal, Murine-Derived, Blotting, Western, Flow Cytometry, Humans, Leukemia, Lymphocytic, Chronic, B-Cell metabolism, Phosphorylation drug effects, RNA, Messenger genetics, RNA, Messenger metabolism, RNA, Small Interfering pharmacology, Reverse Transcriptase Polymerase Chain Reaction, Rituximab, Tristetraprolin antagonists & inhibitors, Tristetraprolin genetics, Tumor Cells, Cultured, Antibodies, Monoclonal therapeutic use, Apoptosis drug effects, Leukemia, Lymphocytic, Chronic, B-Cell pathology, Tristetraprolin metabolism
- Published
- 2009
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13. Why is CLL refractory to bortezomib?
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Wickremasinghe RG
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- 2008
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14. p53-mediated apoptosis of CLL cells: evidence for a transcription-independent mechanism.
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Steele AJ, Prentice AG, Hoffbrand AV, Yogashangary BC, Hart SM, Nacheva EP, Howard-Reeves JD, Duke VM, Kottaridis PD, Cwynarski K, Vassilev LT, and Wickremasinghe RG
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- Adult, Aged, Aged, 80 and over, Apoptosis drug effects, Apoptosis genetics, Apoptosis Regulatory Proteins metabolism, Benzothiazoles pharmacology, Chlorambucil pharmacology, Female, Humans, In Vitro Techniques, Leukemia, Lymphocytic, Chronic, B-Cell genetics, Male, Middle Aged, Mitochondria drug effects, Mitochondria metabolism, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins c-bcl-2 metabolism, Toluene analogs & derivatives, Toluene pharmacology, Transcription, Genetic drug effects, Tumor Suppressor Protein p53 antagonists & inhibitors, Vidarabine analogs & derivatives, Vidarabine pharmacology, Apoptosis physiology, Leukemia, Lymphocytic, Chronic, B-Cell metabolism, Leukemia, Lymphocytic, Chronic, B-Cell pathology, Tumor Suppressor Protein p53 metabolism
- Abstract
The p53 protein plays a key role in securing the apoptotic response of chronic lymphocytic leukemia (CLL) cells to genotoxic agents. Transcriptional induction of proapoptotic proteins including Puma are thought to mediate p53-dependent apoptosis. In contrast, recent studies have identified a novel nontranscriptional mechanism, involving direct binding of p53 to antiapoptotic proteins including Bcl-2 at the mitochondrial surface. Here we show that the major fraction of p53 induced in CLL cells by chlorambucil, fludarabine, or nutlin 3a was stably associated with mitochondria, where it binds to Bcl-2. The Puma protein, which was constitutively expressed in a p53-independent manner, was modestly up-regulated following p53 induction. Pifithrin alpha, an inhibitor of p53-mediated transcription, blocked the up-regulation of Puma and also of p21(CIP1). Surprisingly, pifithrin alpha dramatically augmented apoptosis induction by p53-elevating agents and also accelerated the proapoptotic conformation change of the Bax protein. These data suggest that direct interaction of p53 with mitochondrial antiapoptotic proteins including Bcl-2 is the major route for apoptosis induction in CLL cells and that p53's transcriptional targets include proteins that impede this nontranscriptional pathway. Therefore, strategies that block up-regulation of p53-mediated transcription may be of value in enhancing apoptosis induction of CLL cells by p53-elevating drugs.
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- 2008
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15. The sesquiterpene lactone parthenolide induces selective apoptosis of B-chronic lymphocytic leukemia cells in vitro.
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Steele AJ, Jones DT, Ganeshaguru K, Duke VM, Yogashangary BC, North JM, Lowdell MW, Kottaridis PD, Mehta AB, Prentice AG, Hoffbrand AV, and Wickremasinghe RG
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- Cell Line, Tumor, Dose-Response Relationship, Drug, Drug Screening Assays, Antitumor, Hematopoietic Stem Cells drug effects, Humans, Mitochondria drug effects, Mitochondria metabolism, NF-kappa B drug effects, T-Lymphocytes drug effects, Tumor Suppressor Protein p53 drug effects, Tumor Suppressor Protein p53 metabolism, Up-Regulation, Apoptosis drug effects, Lactones pharmacology, Leukemia, Lymphocytic, Chronic, B-Cell drug therapy, Sesquiterpenes pharmacology
- Abstract
We have studied the in vitro actions of the sesquiterpene lactone parthenolide (PTL) on cells isolated from patients with chronic lymphocytic leukemia (CLL). Dye reduction viability assays showed that the median LD(50) for PTL was 6.2 muM (n=78). Fifteen of these isolates were relatively resistant to the conventional agent chlorambucil but retained sensitivity to PTL. Brief exposures to PTL (1-3 h) were sufficient to induce caspase activation and commitment to cell death. Chronic lymphocytic leukemia cells were more sensitive towards PTL than were normal T lymphocytes or CD34(+) haematopoietic progenitor cells. The mechanism of cell killing was via PTL-induced generation of reactive oxygen species, resulting in turn in a proapoptotic Bax conformational change, release of mitochondrial cytochrome c and caspase activation. Parthenolide also decreased nuclear levels of the antiapoptotic transcription factor nuclear factor-kappa B and diminished phosphorylation of its negative regulator IkappaB. Killing of CLL cells by PTL was apparently independent of p53 induction. This is the first report showing the relative selectivity of PTL towards CLL cells. The data here warrant further investigation of this class of natural product as potential therapeutic agents for CLL.
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- 2006
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16. Ligation of CD8alpha on human natural killer cells prevents activation-induced apoptosis and enhances cytolytic activity.
- Author
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Addison EG, North J, Bakhsh I, Marden C, Haq S, Al-Sarraj S, Malayeri R, Wickremasinghe RG, Davies JK, and Lowdell MW
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- CD8 Antigens blood, Cell Adhesion Molecules blood, Granzymes, HLA Antigens immunology, Humans, Immunophenotyping, Membrane Glycoproteins blood, Perforin, Pore Forming Cytotoxic Proteins, Receptors, Immunologic blood, Receptors, Immunologic immunology, Receptors, KIR, Serine Endopeptidases blood, Signal Transduction immunology, Tumor Cells, Cultured, Apoptosis immunology, CD8 Antigens immunology, Cytotoxicity, Immunologic immunology, Killer Cells, Natural immunology
- Abstract
It has been previously shown that the subset of human natural killer (NK) cells which express CD8 in a homodimeric alpha/alpha form are more cytotoxic than their CD8- counterparts but the mechanisms behind this differential cytolytic activity remained unknown. Target cell lysis by CD8- NK cells is associated with high levels of effector cell apoptosis, which is in contrast to the significantly lower levels found in the CD8alpha+ cells after lysis of the same targets. We report that cross-linking of the CD8alpha chains on NK cells induces rapid rises in intracellular Ca2+ and increased expression of CD69 at the cell surface by initiating the influx of extracellular Ca2+ ions. We demonstrate that secretion of cytolytic enzymes initiates NK-cell apoptosis from which CD8alpha+ NK cells are protected by an influx of exogenous calcium following ligation of CD8 on the NK-cell surface. This ligation is through interaction with fellow NK cells in the cell conjugate and can occur when the target cells lack major histocompatibility complex (MHC) Class I expression. Protection from apoptosis is blocked by preincubation of the NK cells with anti-MHC Class I antibody. Thus, in contrast to the CD8- subset, CD8alpha+ NK cells are capable of sequential lysis of multiple target cells.
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- 2005
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17. CCK2 gastrin receptor as a potential target for therapy in leukaemia cell lines.
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Stubbs M, Khan K, Wickremasinghe RG, Ganeshaguru K, and Caplin ME
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- Cell Line, Tumor, Cell Proliferation, Dose-Response Relationship, Drug, Gastrins chemistry, Gastrins metabolism, Glycine chemistry, Humans, Immunoblotting, Protein Binding, Protein Precursors chemistry, Receptor, Cholecystokinin B metabolism, Receptors, Cholecystokinin metabolism, U937 Cells, Leukemia therapy, Receptor, Cholecystokinin B physiology
- Abstract
The gastrin CCK2 pathway has been implicated in the development of various cancers including leukaemia. An autocrine or intracrine pathway may exist in the leukaemia cell that is involved in stimulating proliferation. We tested four leukaemia cell lines, KU812, ML-1, MOLT-4 and U937 for the existence of the CCK2 receptor and gastrin precursor protein using immunoblotting. We also assessed the effect of CCK2 antagonist PD 135 and both gastrin 17 and glycine-extended gastrin on the proliferation of the cell lines. We found immunoreactive CCK2 and gastrin precursors present in all 4 cell lines. We also observed a stimulatory effect on proliferation by gastrin and glycine-extended gastrin on 2 and 3 of the cell lines respectively and an inhibitory effect of PD 135 on all 4 cell lines. These results demonstrate that the gastrin-gastrin receptor axis is a potential target for new therapeutic strategies.
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- 2005
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18. Selective apoptotic killing of malignant hemopoietic cells by antibody-targeted delivery of an amphipathic peptide.
- Author
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Marks AJ, Cooper MS, Anderson RJ, Orchard KH, Hale G, North JM, Ganeshaguru K, Steele AJ, Mehta AB, Lowdell MW, and Wickremasinghe RG
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- Acute Disease, Antibodies, Monoclonal immunology, Antibodies, Monoclonal metabolism, Antigens, CD immunology, Antigens, CD19 immunology, Antigens, Differentiation, Myelomonocytic immunology, B-Lymphocytes drug effects, Cell Line, Tumor, Drug Screening Assays, Antitumor, Hematologic Neoplasms immunology, Humans, Leukemia, Lymphocytic, Chronic, B-Cell drug therapy, Leukemia, Lymphocytic, Chronic, B-Cell immunology, Leukemia, Myeloid drug therapy, Leukemia, Myeloid immunology, Lymphoma drug therapy, Lymphoma immunology, Sialic Acid Binding Ig-like Lectin 3, Apoptosis drug effects, Hematologic Neoplasms drug therapy, Immunotoxins pharmacology, Peptides administration & dosage
- Abstract
The alpha-helical amphipathic peptide D-(KLAKLAK)2 is toxic to eukaryotic cells if internalized by a suitable targeting mechanism. We have targeted this peptide to malignant hemopoietic cells via conjugation to monoclonal antibodies, which recognize lineage-specific cell surface molecules. An anti-CD19/peptide conjugate efficiently killed 3/3 B lymphoid lines. However, an anti-CD33/peptide conjugate was cytotoxic to only one of three CD33-positive myeloid leukemia lines. The IC50 towards susceptible lines were in the low nanomolar range. Conjugates were highly selective and did not kill cells that did not express the appropriate cell surface cognate of the antibody moiety. Anti-CD19/peptide conjugates efficiently killed cells from patients with chronic lymphocytic leukemia but anti-CD33/peptide reagents were less effective against fresh acute myeloid leukemia cells. We therefore suggest that amphipathic peptides may be of value as targeted therapeutic agents for the treatment of a subset of hematologic malignancies.
- Published
- 2005
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19. Geldanamycin and herbimycin A induce apoptotic killing of B chronic lymphocytic leukemia cells and augment the cells' sensitivity to cytotoxic drugs.
- Author
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Jones DT, Addison E, North JM, Lowdell MW, Hoffbrand AV, Mehta AB, Ganeshaguru K, Folarin NI, and Wickremasinghe RG
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- Antibiotics, Antineoplastic pharmacology, Antigens, CD34 biosynthesis, Benzoquinones, Blotting, Western, Bone Marrow Cells cytology, Cell Separation, Chlorambucil pharmacology, Down-Regulation, Enzyme Inhibitors pharmacology, Flow Cytometry, HSP70 Heat-Shock Proteins biosynthesis, Humans, Inhibitory Concentration 50, Lactams, Macrocyclic, Polymerase Chain Reaction, Protein-Tyrosine Kinases metabolism, RNA, Messenger metabolism, Rifabutin pharmacology, T-Lymphocytes metabolism, Time Factors, Tumor Suppressor Protein p53 metabolism, Up-Regulation, Vidarabine pharmacology, ZAP-70 Protein-Tyrosine Kinase, Apoptosis, Leukemia, Lymphocytic, Chronic, B-Cell drug therapy, Leukemia, Lymphocytic, Chronic, B-Cell pathology, Quinones pharmacology, Vidarabine analogs & derivatives
- Abstract
We studied the actions of geldanamycin (GA) and herbimycin A (HMA), inhibitors of the chaperone proteins Hsp90 and GRP94, on B chronic lymphocytic leukemia (CLL) cells in vitro. Both drugs induced apoptosis of the majority of CLL isolates studied. Whereas exposure to 4-hour pulses of 30 to 100 nM GA killed normal B lymphocytes and CLL cells with similar dose responses, T lymphocytes from healthy donors as well as those present in the CLL isolates were relatively resistant. GA, but not HMA, showed a modest cytoprotective effect toward CD34+ hematopoietic progenitors from normal bone marrow. The ability of bone marrow progenitors to form hematopoietic colonies was unaffected by pulse exposures to GA. Both GA and HMA synergized with chlorambucil and fludarabine in killing a subset of CLL isolates. GA- and HMA-induced apoptosis was preceded by the up-regulation of the stress-responsive chaperones Hsp70 and BiP. Both ansamycins also resulted in down-regulation of Akt protein kinase, a modulator of cell survival. The relative resistance of T lymphocytes and of CD34+ bone marrow progenitors to GA coupled with its ability to induce apoptosis following brief exposures and to synergize with cytotoxic drugs warrant further investigation of ansamycins as potential therapeutic agents in CLL.
- Published
- 2004
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20. Cytotoxic drugs enhance the ex vivo sensitivity of malignant cells from a subset of acute myeloid leukaemia patients to apoptosis induction by tumour necrosis factor receptor-related apoptosis-inducing ligand.
- Author
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Jones DT, Ganeshaguru K, Mitchell WA, Foroni L, Baker RJ, Prentice HG, Mehta AB, and Wickremasinghe RG
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- Acute Disease, Apoptosis drug effects, Apoptosis Regulatory Proteins, Blotting, Western, Caspase 3, Caspases metabolism, Cytarabine administration & dosage, Daunorubicin administration & dosage, Drug Interactions, Humans, Leukemia, Myeloid pathology, Membrane Glycoproteins administration & dosage, TNF-Related Apoptosis-Inducing Ligand, Tumor Cells, Cultured, Tumor Necrosis Factor-alpha administration & dosage, Vidarabine administration & dosage, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Leukemia, Myeloid drug therapy, Membrane Glycoproteins therapeutic use, Tumor Necrosis Factor-alpha therapeutic use, Vidarabine analogs & derivatives
- Abstract
We have studied the actions of tumour-necrosis-factor-related apoptosis-inducing ligand (TRAIL) on cells isolated from patients with acute myeloid leukaemia (AML). Apoptosis induction was initially assessed by quantitative morphological analysis. Only 2/19 isolates showed a > 10% increase in apoptotic cells following TRAIL treatment. However, incubation with TRAIL combined with fludarabine, cytosine arabinoside or daunorubicin resulted in additive or super-additive apoptosis induction in approximately half of the isolates. Molecular evidence of super-additive apoptosis induction by TRAIL and cytotoxic agents was obtained by quantification of caspase 3 activation, detected by Western blot analysis of poly (ADP ribose) polymerase cleavage. The ability of TRAIL and daunorubicin to induce super-additive apoptosis correlated with the ability of these agents to activate caspase 8 and to augment cellular levels of the truncated pro-apoptotic form of the BCL-2 family member BID. Our data suggest that co-administration of TRAIL with conventional cytotoxic drugs may be of therapeutic value in some patients with AML.
- Published
- 2003
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21. Albumin activates the AKT signaling pathway and protects B-chronic lymphocytic leukemia cells from chlorambucil- and radiation-induced apoptosis.
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Jones DT, Ganeshaguru K, Anderson RJ, Jackson TR, Bruckdorfer KR, Low SY, Palmqvist L, Prentice HG, Hoffbrand AV, Mehta AB, and Wickremasinghe RG
- Subjects
- Apoptosis drug effects, Apoptosis radiation effects, Chlorambucil pharmacology, Chromones pharmacology, Endocytosis, Enzyme Inhibitors pharmacology, Gamma Rays, Humans, Microscopy, Confocal, Morpholines pharmacology, Phosphatidylinositol 3-Kinases physiology, Phosphoinositide-3 Kinase Inhibitors, Proto-Oncogene Proteins c-akt, Radiation-Protective Agents pharmacology, Recombinant Proteins pharmacology, Tumor Cells, Cultured drug effects, Tumor Cells, Cultured radiation effects, Leukemia, Lymphocytic, Chronic, B-Cell pathology, Protein Serine-Threonine Kinases, Proto-Oncogene Proteins physiology, Serum Albumin pharmacology, Signal Transduction drug effects
- Abstract
Activation of the phosphatidylinositol 3- kinase/AKT pathway antagonizes apoptosis in diverse cellular systems. We previously showed that human plasma activated AKT and potently blocked the ability of chlorambucil or gamma radiation to induce apoptosis of B-chronic lymphocytic leukemia (CLL) cells. Here we report experiments that identify albumin as the major component of plasma that blocks CLL cell killing by chlorambucil or radiation. Intact plasma depleted of albumin by chromatography on Cibacron blue-Sepharose or plasma from a subject with analbuminemia failed either to activate AKT or to protect CLL cells from chlorambucil-induced apoptosis. Both functions were restored by re-addition of albumin. The protective action of albumin as well as AKT activation was compromised by the binding of lipids. Fluorescence-activated cell sorter (FACScan) analysis demonstrated the uptake of fluoresceinated albumin by CLL cells. Accumulation of albumin in intracellular vesicles was also shown by confocal microscopy. Indirect inhibition of AKT activation by the phosphatidylinositol 3-kinase inhibitor LY294002 reversed the blockade of chlorambucil-induced killing by plasma albumin. The data suggest that activation of AKT consequent to binding of albumin by CLL cells blocks chlorambucil- and radiation-induced apoptosis. Strategies designed to block albumin-induced antiapoptotic signaling may, therefore, be of value in enhancing cytotoxic drug action on CLL cells.
- Published
- 2003
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22. Actions of the selective protein kinase C inhibitor PKC412 on B-chronic lymphocytic leukemia cells in vitro.
- Author
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Ganeshaguru K, Wickremasinghe RG, Jones DT, Gordon M, Hart SM, Virchis AE, Prentice HG, Hoffbrand AV, Man A, Champain K, Csermak K, and Mehta AB
- Subjects
- 2-Chloroadenosine pharmacology, ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism, Aged, Aged, 80 and over, Apoptosis drug effects, B-Lymphocytes drug effects, B-Lymphocytes enzymology, Calcium Channel Blockers pharmacology, Chlorambucil pharmacology, Deoxyadenosines pharmacology, Drug Resistance, Multiple, Drug Resistance, Neoplasm, Drug Screening Assays, Antitumor, Female, Humans, In Situ Nick-End Labeling, Male, Middle Aged, Palatine Tonsil cytology, Tumor Cells, Cultured drug effects, Tumor Cells, Cultured enzymology, Verapamil pharmacology, Vidarabine analogs & derivatives, Vidarabine pharmacology, 2-Chloroadenosine analogs & derivatives, Antineoplastic Agents pharmacology, Enzyme Inhibitors pharmacology, Leukemia, Lymphocytic, Chronic, B-Cell pathology, Neoplasm Proteins antagonists & inhibitors, Protein Kinase C antagonists & inhibitors, Staurosporine analogs & derivatives, Staurosporine pharmacology
- Abstract
Background and Objectives: The staurosporine derivative PKC412 (CGP41251) is a more selective inhibitor of the conventional isoforms of protein kinase C (PKC) than is the parent compound. In addition to its growth inhibitory properties, PKC412 reverses the efflux function of the multidrug resistance (MDR)-1 gene product, P-glycoprotein (P-gp)., Design and Methods: The in vitro actions of PKC412 were investigated in peripheral blood lymphocytes (PBL) from 4 normal volunteers, B-cell isolates from 3 normal tonsils and 31 patients with B-cell chronic lymphocytic leukemia (B-CLL). Following incubation with PKC412 for 2 days, the viability of B-CLL cells was decreased relative to that of controls (63+/-23% at 1 micromole/L; 52+/-30% at 10 micromole/L; n=20). Normal PBL were significantly more resistant to the drug (91+/-5% viable cells at 1 micromole/L; 73+/-18% at 10 micromole/L; n=4). Thirteen of the B-CLL patients were treated with oral PKC412 in a phase II trial., Results: PKC activity in malignant cells from these patients showed a reduction post-treatment of 25-96% of their respective pre-treatment levels. Morphologic analysis, as well as in situ assay for DNA strand breaks (TUNEL assay) showed that B-CLL cells were killed by an apoptotic mechanism. In B-CLL cells the mean IC50, for PKC412, as measured by the reduction of 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), was 2.1 micromol/L in 16 samples in which the IC50 were below the maximum concentration of PKC412 used for the assay. In tonsillar B-cells, the mean IC50 was 11 micromol/L whereas PBL cells were resistant. Four of eight and 1/3 B-CLL samples that were resistant to chlorambucil and fludarabine, respectively, were sensitive to PKC412. In 15/31 B-CLL samples a dose-dependent reversal of P-gp-mediated drug efflux by PKC412 was observed. A statistically significant correlation (p<0.001) was observed between P-gp protein expression as measured by FACScan analysis and the reversal of efflux activity by either PKC412 or verapamil. PKC412 increased the sensitivity of B-CLL cells to 2'-chlorodeoxyadenosine and chlorambucil., Interpretation and Conclusions: This study establishes the in vitro cytotoxic and multidrug resistance (MDR) modulatory properties of PKC412 towards malignant cells from B-CLL patients. The direct antitumor activity combined with the potential for P-gp modulation make PKC412 an attractive drug for the treatment of malignancies expressing the MDR phenotype, or in combination with conventional drugs.
- Published
- 2002
23. Caspase 8 activation independent of Fas (CD95/APO-1) signaling may mediate killing of B-chronic lymphocytic leukemia cells by cytotoxic drugs or gamma radiation.
- Author
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Jones DT, Ganeshaguru K, Virchis AE, Folarin NI, Lowdell MW, Mehta AB, Prentice HG, Hoffbrand AV, and Wickremasinghe RG
- Subjects
- Antibodies, Monoclonal pharmacology, Antineoplastic Agents pharmacology, Apoptosis physiology, B-Lymphocytes drug effects, B-Lymphocytes pathology, B-Lymphocytes radiation effects, Caspase 8, Caspase 9, Caspases metabolism, Drug Interactions, Enzyme Activation, Fas Ligand Protein, Female, Gamma Rays, Humans, Leukemia, Lymphocytic, Chronic, B-Cell drug therapy, Leukemia, Lymphocytic, Chronic, B-Cell radiotherapy, Male, Membrane Glycoproteins analysis, fas Receptor analysis, fas Receptor immunology, Apoptosis drug effects, Caspases pharmacology, Leukemia, Lymphocytic, Chronic, B-Cell pathology, Signal Transduction, fas Receptor pharmacology
- Abstract
Ligation of the cell-surface Fas molecule by its ligand (Fas-L) or agonistic anti-Fas monoclonal antibodies results in the cleavage and activation of the cysteine protease procaspase 8 followed by the activation of procaspase 3 and by apoptosis. In some leukemia cell lines, cytotoxic drugs induce expression of Fas-L, which may contribute to cell killing through the ligation of Fas. The involvement of Fas, Fas-L, and caspase 8 was studied in the killing of B-cell chronic lymphocytic leukemia (B-CLL) cells by chlorambucil, fludarabine, or gamma radiation. Spontaneous apoptosis was observed at 24-hour incubation, with additional apoptosis induced by each of the cytotoxic treatments. Although Fas mRNA expression was elevated after exposure to chlorambucil, fludarabine, or gamma radiation, Fas protein levels only increased after irradiation. Therefore, Fas expression may be regulated by multiple mechanisms that allow the translation of Fas mRNA only in response to restricted cytotoxic stimuli. None of the cytotoxic stimuli studied here induced Fas-L expression. An agonistic anti-Fas monoclonal antibody (CH-11) did not significantly augment apoptosis induction by any of the death stimuli. A Fas-blocking antibody (ZB4) did not inhibit spontaneous, chlorambucil-, fludarabine-, or radiation-induced apoptosis. However, procaspase 8 processing was induced by all cytotoxic stimuli. These data suggest that the Fas/Fas-L signaling system does not play a major role in the induction of apoptosis in B-CLL cells treated with cytotoxic drugs or radiation. However, Fas-independent activation of caspase 8 may play a crucial role in the regulation of apoptosis in these cells.
- Published
- 2001
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24. Autologous plasma activates Akt/protein kinase B and enhances basal survival and resistance to DNA damage-induced apoptosis in B-chronic lymphocytic leukaemia cells.
- Author
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Wickremasinghe RG, Ganeshaguru K, Jones DT, Lindsay C, Spanswick VJ, Hartley JA, Wadhwa M, Thorpe R, Hoffbrand AV, Prentice HG, and Mehta AB
- Subjects
- Antineoplastic Agents, Alkylating therapeutic use, Apoptosis drug effects, Apoptosis radiation effects, Blotting, Western, Chlorambucil therapeutic use, Chromones pharmacology, Enzyme Inhibitors pharmacology, Female, Humans, Interleukin-4 therapeutic use, Leukemia, Lymphocytic, Chronic, B-Cell drug therapy, Male, Morpholines pharmacology, Phosphoinositide-3 Kinase Inhibitors, Proto-Oncogene Proteins c-akt, Tumor Cells, Cultured, Drug Resistance, Enzyme Activation, Leukemia, Lymphocytic, Chronic, B-Cell enzymology, Plasma, Protein Serine-Threonine Kinases metabolism, Proto-Oncogene Proteins
- Abstract
We have studied the actions of autologous plasma on both basal and DNA damage-induced apoptosis in B-chronic lymphocytic leukaemia (B-CLL) cells. Apoptosis was quantified using morphological criteria and Western blot analysis for the apoptosis-specific p85 fragment of poly(ADP ribose) polymerase. Cell viability was estimated using the methyl thiazol tetrazolium bromide dye reduction assay. Plasma cultures showed lower rates of basal apoptosis as well as a decreased cytotoxic response to chlorambucil and gamma-radiation compared with cultures in fetal calf serum. Experiments using neutralizing antibodies suggested that the protective actions of plasma could not be accounted for by interleukin 4, the interferons alpha or gamma or stromal cell-derived factor 1, each of which have been shown to protect B-CLL cells from apoptosis in vitro. Plasma addition to B-CLL cells resulted in rapid activation of the Akt protein kinase, a key signalling enzyme that has been implicated in anti-apoptotic signalling. LY294002, an inhibitor of phosphatidylinositol 3'-kinase, blocked Akt activation by plasma. To the best of our knowledge, this is the first report to show that factors present in plasma promote basal survival of B-CLL cells and resistance to cytotoxic drugs via stimulation of the Akt cytoprotective-signalling pathway. Pharmacological blockade of this pathway may have potential in the development of novel therapeutic strategies for B-CLL treatment.
- Published
- 2001
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25. Antioxidants enhance the susceptibility of colon carcinoma cells to 5-fluorouracil by augmenting the induction of the bax protein.
- Author
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Adeyemo D, Imtiaz F, Toffa S, Lowdell M, Wickremasinghe RG, and Winslet M
- Subjects
- Acetylcysteine pharmacology, Antimetabolites, Antineoplastic therapeutic use, Apoptosis, Blotting, Western, Drug Interactions, Flow Cytometry, Free Radical Scavengers pharmacology, Humans, Oxidation-Reduction, Reactive Oxygen Species, Time Factors, Tumor Cells, Cultured, Vitamin E pharmacology, bcl-2-Associated X Protein, Antioxidants therapeutic use, Colonic Neoplasms drug therapy, Colonic Neoplasms genetics, Fluorouracil therapeutic use, Proto-Oncogene Proteins biosynthesis, Proto-Oncogene Proteins c-bcl-2
- Abstract
5 Fluorouracil (5 FU), the most effective systemic chemotherapeutic agent in the management of advanced colorectal carcinoma acts by inducing apoptosis. Response rates, approximately 20% is improved by folinic acid. This study investigates similar modulation of 5 FU-induced apoptosis by oxidant quenching. A five-fold reduction of intracellular oxidant levels by antioxidants N-acetylcysteine and vitamin E did not induce apoptosis, it however augmented pro-apoptotic bax protein expression, and apoptotic response to a non-toxic dose of 5 FU in the colorectal cancer cell lines colo 201 and colo 205. This suggests that reduction of intracellular levels of reactive oxygen species enhance susceptibility to 5 FU (apoptotic stimuli) by augmentation of bax expression.
- Published
- 2001
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26. Biochemical and genetic control of apoptosis: relevance to normal hematopoiesis and hematological malignancies.
- Author
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Wickremasinghe RG and Hoffbrand AV
- Subjects
- Animals, Humans, Proto-Oncogene Proteins c-bcl-2 genetics, Tumor Suppressor Protein p53 genetics, Apoptosis genetics, Gene Expression Regulation, Neoplastic, Hematologic Neoplasms genetics, Hematologic Neoplasms pathology, Hematopoiesis genetics
- Published
- 1999
27. Killing of T lymphocytes by synthetic ceramide is by a nonapoptotic mechanism and is abrogated following mitogenic activation.
- Author
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Mengubas K, Fahey AA, Lewin J, Mehta AB, Hoffbrand AV, and Wickremasinghe RG
- Subjects
- Amino Acid Chloromethyl Ketones pharmacology, Apoptosis drug effects, Caspases metabolism, Cell Death, Cells, Cultured, Cysteine Proteinase Inhibitors pharmacology, Enzyme Activation drug effects, Humans, Jurkat Cells drug effects, Lymphocyte Activation drug effects, Mitogens pharmacology, Neoplastic Stem Cells drug effects, Neoplastic Stem Cells pathology, Okadaic Acid pharmacology, Phytohemagglutinins pharmacology, Sphingosine chemical synthesis, Sphingosine pharmacology, Sphingosine analogs & derivatives, T-Lymphocytes drug effects
- Abstract
Ceramide induces apoptosis in leukemia cell lines and has been proposed as a potential therapeutic agent in malignancies refractory to conventional treatment. Here we show that synthetic N-acetyl-d-erythro-sphingosine (C2 ceramide) kills normal human T lymphocytes by a caspase-independent nonapoptotic mechanism. By contrast, T cells were induced to caspase-dependent apoptosis by okadaic acid. Furthermore, C2 ceramide treatment of the Jurkat leukemia cell line induced killing by apoptosis. Activation of T lymphocytes by phytohemagglutinin abrogated killing by C2 ceramide. The data here suggest that ceramide triggers caspase-dependent apoptosis in leukemia cells lines, but activates caspase-independent nonapoptotic killing of resting T lymphocytes which is abrogated following mitogenic activation., (Copyright 1999 Academic Press.)
- Published
- 1999
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28. Ceramide-induced killing of normal and malignant human lymphocytes is by a non-apoptotic mechanism.
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Mengubas K, Riordan FA, Bravery CA, Lewin J, Owens DL, Mehta AB, Hoffbrand AV, and Wickremasinghe RG
- Subjects
- Amidohydrolases antagonists & inhibitors, Antineoplastic Agents pharmacology, Apoptosis, Caspase 3, Caspases drug effects, Caspases metabolism, Cell Death drug effects, Ceramidases, Enzyme Inhibitors pharmacology, Gamma Rays, Humans, Leukemia, Lymphocytic, Chronic, B-Cell pathology, Leukemia, Lymphocytic, Chronic, B-Cell radiotherapy, Lymphocytes radiation effects, Myristates pharmacology, Propanolamines pharmacology, Protein Biosynthesis, Proteins drug effects, Proto-Oncogene Proteins drug effects, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins c-bcl-2 drug effects, Proto-Oncogene Proteins c-bcl-2 genetics, Proto-Oncogene Proteins c-bcl-2 metabolism, Sphingosine pharmacology, Vidarabine analogs & derivatives, Vidarabine pharmacology, bcl-2-Associated X Protein, Leukemia, Lymphocytic, Chronic, B-Cell drug therapy, Lymphocytes drug effects, Lymphocytes pathology, Sphingosine analogs & derivatives
- Abstract
Synthetic ceramides induce apoptotic death of Jurkat and HL60 leukaemia cell lines. By contrast we show here that ceramide induces non-apoptotic killing of malignant cells from patients with B-chronic lymphocytic leukaemia (B-CLL) and of normal B lymphocytes. The protein phosphatase inhibitor okadaic acid readily induces apoptosis of B-CLL cells, indicating that this death pathway is fully functional in these cells. The ability of ceramide to activate the apoptotic protease caspase 3 in HL60 cells but not in B-CLL cells, as well as the lack of correlation of ceramide-mediated killing of different B-CLL isolates with expression of the apoptosis-regulating proteins bcl-2 and bax reinforce the conclusion that ceramide killing of B-CLL cells is by a non-apoptotic mechanism. Fludarabine treatment or gamma-irradiation of B-CLL cells resulted in ceramide elevation and in killing by both apoptotic and non-apoptotic mechanisms, suggesting that a ceramide-triggered non-apoptotic mechanism may play a role in the killing of these cells. Therefore, the results here show that ceramide can induce either apoptotic or non-apoptotic death, depending on the cellular context. The inability of synthetic dihydroceramide to kill B-CLL cells or normal B lymphocytes suggests that non-apoptotic killing by ceramide is via interaction with a specific, but unidentified, cellular target.
- Published
- 1999
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29. Activation-associated necrosis in human immunodeficiency virus infection.
- Author
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Borthwick NJ, Wickremasinghe RG, Lewin J, Fairbanks LD, and Bofill M
- Subjects
- Adult, Apoptosis, CD3 Complex analysis, CD3 Complex immunology, CD5 Antigens analysis, CD5 Antigens immunology, Cell Count, Cytokines immunology, Cytokines pharmacology, HIV Infections immunology, Humans, Lymphocytes immunology, Male, Middle Aged, Necrosis, Nucleotides biosynthesis, HIV Infections pathology, Lymphocyte Activation, Lymphocytes pathology
- Abstract
Mitogenic stimulation of lymphocytes from persons infected with human immunodeficiency virus (HIV) resulted in massive cell death. In addition to early apoptosis, a second wave of cell death occurred 48-72 h after stimulation. At that time, the cells were enlarged, leaked content, and had plasma membrane damage-all indicative of necrosis. Furthermore, DNA fragmentation as determined by TUNEL assay was virtually absent. This activation-associated necrosis could not be prevented by interfering with CD95/CD95-ligand interactions or by blocking caspase activity and was unaffected by neutralizing antibodies to tumor necrosis factor-alpha or interferon-gamma. Necrosis was also induced by activation of normal lymphocytes in the presence of ribavirin, which inhibits the de novo pathway of nucleotide synthesis. Lymphocytes from HIV-infected persons are defective in their ability to synthesize nucleotides via this pathway, indicating one possible mechanism for the activation-associated necrosis seen in HIV infection.
- Published
- 1999
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30. Monocytes stimulate expression of the Bcl-2 family member, A1, in endothelial cells and confer protection against apoptosis.
- Author
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Noble KE, Wickremasinghe RG, DeCornet C, Panayiotidis P, and Yong KL
- Subjects
- Apoptosis, Base Sequence, Cell Adhesion, Cell Adhesion Molecules physiology, Cell Communication, Cell Survival, Coculture Techniques, DNA Primers genetics, Gene Expression, Humans, Inflammation etiology, Inflammation pathology, Inflammation physiopathology, Interleukin-1 pharmacology, Interleukin-10 pharmacology, Interleukin-10 physiology, Minor Histocompatibility Antigens, Monocytes immunology, RNA, Messenger genetics, RNA, Messenger metabolism, Replication Protein C, Signal Transduction, Tumor Necrosis Factor-alpha pharmacology, Tumor Necrosis Factor-alpha physiology, DNA-Binding Proteins genetics, Endothelium, Vascular cytology, Endothelium, Vascular physiology, Homeodomain Proteins, Monocytes physiology, Proto-Oncogene Proteins c-bcl-2 genetics, Repressor Proteins, Saccharomyces cerevisiae Proteins
- Abstract
We have investigated the molecular mechanisms underlying the ability of peripheral blood monocytes to block apoptosis induction in endothelial cells. Monocytes stimulated the expression of the bcl-2 homologue A1 in serum-starved endothelial cells after 6 h of coincubation, with elevated A1 levels persisting for up to 21 h. IL-1 and TNF also stimulated A1 expression at 6 h, but A1 transcript levels fell by 21 h. Direct cellular contact with monocytes was required for stimulation of A1 mRNA in endothelial cells. Stimulation of endothelial cell A1 mRNA by monocytes was not inhibited by anti-beta2 integrin Abs, but anti-platelet endothelial cell adhesion molecule-1 (PECAM-1) mAb reduced A1 transcript levels at 21 h. Studies employing either TNF on its own, or anti-TNF in endothelium/monocyte cocultures showed that TNF plays a role in the early (6-h) stimulation of A1, but is less important for the sustained elevation of A1 levels at 21 h. Serum-starved endothelial cells demonstrated increased survival and decreased apoptosis after coculture with monocytes. IL-10 reduced A1 mRNA expression in, as well as survival of, endothelial cells that were cocultured with monocytes. In comparison with A1, Bcl-2 was expressed at low levels and was up-regulated by monocytes only at 21 h, while neither Bax nor Bcl-xL levels were altered by monocytes. The interaction of monocytes with endothelium during the course of an inflammatory reaction may provide survival signals to endothelial cells.
- Published
- 1999
31. Integrin signalling defects in T-lymphocytes in systemic lupus erythematosus.
- Author
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Ng TT, Collins IE, Kanner SB, Humphries MJ, Amft N, Wickremasinghe RG, D'Cruz D, Nye KE, and Morrow WJ
- Subjects
- Adolescent, Adult, Aged, Antibodies, Antinuclear biosynthesis, Cell Adhesion, Enzyme Activation, Female, Humans, Integrin beta1 analysis, Isoenzymes metabolism, Lymphocyte Activation, Male, Middle Aged, Phosphorylation, Protein Kinase C metabolism, T-Lymphocytes physiology, Integrin beta1 physiology, Lupus Erythematosus, Systemic immunology, T-Lymphocytes immunology
- Abstract
Objective: To establish the relationship between T cell responses to integrin coreceptor stimulation and B cell hyperreactivity as measured by pathologic autoantibody production., Methods: Peripheral blood mononuclear cells from 42 patients with SLE according to the American Rheumatism Association criteria were examined for their ability to adhere to plate-immobilised fibronectin. Co-stimulation assays were performed on the same cells using anti-CD3 antibody alone or co-immobilised with an anti-beta1-integrin antibody. Proliferative responses were measured by 3[H]thymidine pulsing on day 3 and activation was determined using a commercial protein kinase C assay, the protocol being established by our group in association with Promega. Beta-integrin expression was established by FACS analysis., Results: An impaired PKC response to integrin-mediated activation was found in T-lymphocytes from 6/21 (29%) SLE patients, which correlated significantly with an absence of anti-dsDNA antibody in patient sera, irrespective of prednisolone treatment. Integrin co-stimulation of TcR/CD3-induced proliferation and T cell adhesion to fibronectin were also impaired among 5/21 (24%) and 6/15 (40%) patients studied, respectively., Conclusion: We hypothesise that the integrity of beta1-integrin signalling pathways may influence pathological antibody production in SLE by affecting T-lymphocyte activation and interactions between T- and B-lymphocytes.
- Published
- 1999
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32. Okadaic acid-induced apoptosis of HL60 leukemia cells is preceded by destabilization of bcl-2 mRNA and downregulation of bcl-2 protein.
- Author
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Riordan FA, Foroni L, Hoffbrand AV, Mehta AB, and Wickremasinghe RG
- Subjects
- Down-Regulation, HL-60 Cells, Humans, Phosphoprotein Phosphatases antagonists & inhibitors, Proto-Oncogene Proteins c-bcl-2 genetics, RNA, Messenger biosynthesis, Apoptosis drug effects, Enzyme Inhibitors pharmacology, Okadaic Acid pharmacology, Proto-Oncogene Proteins c-bcl-2 biosynthesis
- Abstract
We have studied the actions of the protein phosphatase inhibitor okadaic acid (OA) on the expression of bcl-2 in HL60 human leukemia cells. OA induced downregulation of bcl-2 mRNA and protein prior to the induction of apoptosis. Downregulation of bcl-2 mRNA levels did not result from actions of OA on the bcl-2 upstream negative response element. Nuclear run-off analyses confirmed that OA did not affect bcl-2 gene transcription. However, OA caused a rapid increase in the rate of degradation of bcl-2 mRNA. Therefore, OA induces down-regulation of bcl-2 expression via destabilization of its transcript. The constitutive action of an OA-sensitive protein phosphatase may therefore maintain HL60 cell survival by blocking bcl-2 mRNA degradation.
- Published
- 1998
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33. Herbimycin A accelerates the induction of apoptosis following etoposide treatment or gamma-irradiation of bcr/abl-positive leukaemia cells.
- Author
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Riordan FA, Bravery CA, Mengubas K, Ray N, Borthwick NJ, Akbar AN, Hart SM, Hoffbrand AV, Mehta AB, and Wickremasinghe RG
- Subjects
- Antibiotics, Antineoplastic pharmacology, Antineoplastic Agents, Phytogenic pharmacology, Apoptosis genetics, Benzoquinones, Fusion Proteins, bcr-abl drug effects, Fusion Proteins, bcr-abl radiation effects, HL-60 Cells, Humans, Lactams, Macrocyclic, Leukemia, Erythroblastic, Acute genetics, Leukemia, Myelogenous, Chronic, BCR-ABL Positive drug therapy, Leukemia, Myelogenous, Chronic, BCR-ABL Positive pathology, Proto-Oncogene Proteins c-bcl-2 biosynthesis, Proto-Oncogene Proteins c-bcl-2 drug effects, Rifabutin analogs & derivatives, Tumor Cells, Cultured, bcl-X Protein, Apoptosis drug effects, Apoptosis radiation effects, Etoposide pharmacology, Fusion Proteins, bcr-abl analysis, Gamma Rays, Leukemia, Erythroblastic, Acute drug therapy, Leukemia, Erythroblastic, Acute pathology, Quinones pharmacology
- Abstract
Philadelphia chromosome (Ph)-positive leukaemia cells express the chimeric bcr/abl oncoprotein, whose deregulated protein tyrosine kinase (PTK) activity antagonizes the induction of apoptosis by DNA damaging agents. Treatment of Ph-positive K562, TOM 1 and KCL-22 cells with etoposide for 2d induced cytosolic vacuolation, but not nuclear condensation or DNA fragmentation. The bcr/abl kinase-selective inhibitor herbimycin A increased the induction of nuclear apoptosis by etoposide or gamma-radiation. The concentration of herbimycin required to synergize with etoposide was similar to that required to decrease the level of tyrosine phosphorylated proteins or of the protein tyrosine kinase activity of anti-abl immune complexes in K562 cells. The ability of herbimycin A to sensitize K562, TOM 1 or KCL-22 cells to apoptosis induction correlated with its ability to decrease the cellular content of phosphotyrosyl proteins in these Philadelphia-positive lines. Enhancement of nuclear apoptosis by herbimycin was not attributable to downregulation of the bcl-2 or bcl-XL anti-apoptotic proteins. In contrast, herbimycin protected Philadelphia-negative HL60 cells from apoptosis induction by etoposide and did not affect killing of NC37 and CEM cells. The data suggest that the induction of apoptosis is blocked in cells expressing the bcr/abl oncoprotein and that herbimycin A increases induction of programmed cell death following DNA damage. Selective PTK inhibitors may therefore be of value in securing the genetic death of Ph-positive leukaemia cells.
- Published
- 1998
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34. Signal Transduction by the Philadelphia Chromosome-encoded BCR/ABL Oncoproteins: Therapeutic Implications for Chronic Myeloid Leukemia and Philadelphia-positive Acute Lymphoblastic Leukemia.
- Author
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Riordan FA and Wickremasinghe RG
- Abstract
The Philadelphia chromosomes characteristic of chronic myeloid leukemia (CML) and Philadelphia-positive acute lymphoblastic leukemia (ALL) encode chimeric protein tyrosine kinases (PTKs) derived by fusion of the normal BCR and ABL genes. The oncogenic properties of these BCR/ABL oncoproteins are dependent on their elevated PTK activity and on their ability to interact with multiple signal transduction systems. Here we summarize some of the key pathways which are activated by normal receptors with PTK activity and which modulate cell proliferation and survival. Next, we review some of the biochemical pathways initiated by BCR/ABL oncoproteins and discuss their possible relevance to the leukemic phenotype. We finally review experimental approaches designed to suppress signalling by BCR/ABL oncoproteins and discuss their potential therapeutic applications.
- Published
- 1998
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35. The integrin-triggered rescue of T lymphocyte apoptosis is blocked in HIV-1-infected individuals.
- Author
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Ng TT, Kanner SB, Humphries MJ, Wickremasinghe RG, Nye KE, Anderson J, Khoo SH, and Morrow WJ
- Subjects
- Acquired Immunodeficiency Syndrome immunology, Anti-HIV Agents therapeutic use, Antibodies, Monoclonal pharmacology, Apoptosis drug effects, CD4-Positive T-Lymphocytes immunology, Cell Adhesion Molecules biosynthesis, Cell Adhesion Molecules blood, Cell Adhesion Molecules genetics, Drug Synergism, Enzyme Activation drug effects, Epitopes physiology, Focal Adhesion Kinase 1, Focal Adhesion Protein-Tyrosine Kinases, HIV Infections drug therapy, HIV Infections metabolism, Humans, Immune Tolerance, Integrin beta1 biosynthesis, Integrins metabolism, Interferon-gamma metabolism, Interphase, Leukemia, Lymphoid, Lymphocyte Activation drug effects, Protein Kinase C drug effects, Protein-Tyrosine Kinases biosynthesis, Protein-Tyrosine Kinases blood, Protein-Tyrosine Kinases genetics, RNA, Messenger biosynthesis, Receptor-CD3 Complex, Antigen, T-Cell antagonists & inhibitors, Receptor-CD3 Complex, Antigen, T-Cell biosynthesis, Receptor-CD3 Complex, Antigen, T-Cell physiology, Signal Transduction drug effects, Signal Transduction immunology, T-Lymphocytes drug effects, Tumor Cells, Cultured, Apoptosis immunology, HIV Infections immunology, Integrins physiology, T-Lymphocytes immunology
- Abstract
HIV infection is associated with a disease status-dependent impairment of Ag-specific T cell responses, resulting in anergy or unchecked apoptotic cell death. beta1 integrins play an important role in the induction of T lymphocyte responses to antigenic challenge by providing a T cell costimulatory signal, and have been shown to rescue various cell types from undergoing apoptosis. We examined the integrin-triggered cell survival signal and associated pathways in CD3+ T cells derived from 69 HIV-1-infected individuals in comparison with healthy controls. We found beta1 integrin-mediated costimulation of TCR-induced T cell proliferation and protection from aberrant cell death to be absent in the majority of patients with AIDS, but intact in asymptomatic, infected individuals. The lack of integrin-mediated rescue may be partly due to an early impairment of TCR/integrin-costimulated secretion of IFN-gamma, a type 1 lymphokine that protects against TCR-induced apoptosis of T cells from HIV-seropositive donors, but not loss of integrin expression. The mechanism of integrin hyporesponsiveness appeared to correlate with a failure of the integrin-generated signal to induce pp125FAK mRNA and protein expression. Protein kinase C activation in CD3+ T cells following integrin stimulation was also impaired in HIV-infected individuals, mostly among the symptomatic/AIDS patients. Protein kinase C inactivation in T cells was shown to have a destabilizing effect in vitro on pp125FAK mRNA that contains an AUUUA motif in the 3'-untranslated region, a consensus sequence for the AU-rich elements responsible for mRNA destabilization. These aberrant changes in pp125FAK expression may have direct significance to the overall immunopathogenesis during infection with HIV-1.
- Published
- 1997
36. Co-ordinated downregulation of bcl-2 and bax expression during granulocytic and macrophage-like differentiation of the HL60 promyelocytic leukaemia cell line.
- Author
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Mengubas K, Riordan FA, Hoffbrand AV, and Wickremasinghe RG
- Subjects
- Apoptosis genetics, Cell Differentiation, Down-Regulation, Granulocytes physiology, HL-60 Cells, Humans, Leukemia, Myeloid genetics, Macrophages physiology, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins c-bcl-2 genetics, RNA Splicing, RNA, Messenger metabolism, Tetradecanoylphorbol Acetate pharmacology, Tretinoin pharmacology, bcl-2-Associated X Protein, Gene Expression Regulation, Developmental, Proto-Oncogene Proteins biosynthesis, Proto-Oncogene Proteins c-bcl-2 biosynthesis
- Abstract
The bcl-2 protein suppresses apoptosis and the bax protein opposes the cytoprotective effect of bcl-2. A decrease in bcl-2 levels has been implicated in the induction of apoptosis during the terminal differentiation of HL60 myeloid leukaemia cells. We show here that bax protein also declined with a time course similar to the downregulation of bcl-2 following treatment of HL60 with phorbol myristate acetate (PMA), dimethyl sulphoxide (DMSO) or retinoic acid (RA). Decreased bcl-2 protein expression in induced cells was associated with down-regulation of its mRNA. By contrast, the decrease in bax occurred by a post-transcriptional mechanism. Co-ordinate downregulation of bcl-2 and bax proteins may fine-tune the induction of apoptosis during cellular differentiation.
- Published
- 1996
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37. Interleukin-2 receptor common gamma-chain signaling cytokines regulate activated T cell apoptosis in response to growth factor withdrawal: selective induction of anti-apoptotic (bcl-2, bcl-xL) but not pro-apoptotic (bax, bcl-xS) gene expression.
- Author
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Akbar AN, Borthwick NJ, Wickremasinghe RG, Panayoitidis P, Pilling D, Bofill M, Krajewski S, Reed JC, and Salmon M
- Subjects
- Base Sequence, Cell Line, Cell Survival drug effects, Cell Survival immunology, Cytokines classification, Etoposide pharmacology, Humans, Molecular Sequence Data, Proto-Oncogene Proteins biosynthesis, Proto-Oncogene Proteins c-bcl-2, RNA, Messenger biosynthesis, Receptors, Interleukin-2 chemistry, Signal Transduction drug effects, Up-Regulation drug effects, Up-Regulation genetics, Up-Regulation immunology, bcl-2-Associated X Protein, bcl-X Protein, Apoptosis immunology, Cytokines physiology, Gene Expression Regulation immunology, Growth Substances pharmacology, Lymphocyte Activation genetics, Proto-Oncogene Proteins genetics, Receptors, Interleukin-2 immunology, Signal Transduction immunology, T-Lymphocytes immunology
- Abstract
Cytokine deprivation from activated T cells leads to apoptosis associated with down-regulation of the bcl-2 gene product. It is not clear, however, how cytokines other than interleukin-2 (IL-2) may affect this process and regulate the involvement of other apoptosis-modulating genes. We show that a group of cytokines including IL-2 (IL-2R gamma), prevent the apoptosis of IL-2-deprived activated T cells. This rescue involves the induction of the anti-apoptosis genes bcl-2 and bcl-xL), but causes little change in expression of bax and bcl-xS, which promote apoptosis. Furthermore, the prevention of apoptosis and induction of proliferation by the common gamma chain cytokines can be dissociated. Thus, when proliferation is blocked, the common gamma chain cytokines still induce up-regulation of bcl-2 relative to bax and retard apoptosis. These cytokines can thus regulate the persistence or removal of effector T cells by coordinating the balance between genes which promote and those which inhibit apoptosis, events which are probably mediated at least in part by signals through the common gamma chain. These data also implicate inappropriate T cell apoptosis resulting from a dysfunctional common gamma-chain as part of the pathophysiological defect in patients with X-linked severe-combined immunodeficiency (SCID).
- Published
- 1996
- Full Text
- View/download PDF
38. Deletion of chromosome 13 (band q14) but not trisomy 12 is a clonal event in B-chronic lymphocytic leukaemia (CLL).
- Author
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Jabbar SA, Ganeshaguru K, Wickremasinghe RG, Hoffbrand AV, and Foroni L
- Subjects
- Blotting, Southern, Humans, In Situ Hybridization, Fluorescence, Chromosome Deletion, Chromosomes, Human, Pair 12, Chromosomes, Human, Pair 13, Leukemia, Lymphocytic, Chronic, B-Cell genetics, Trisomy
- Abstract
Chromosomal abnormalities are detected by conventional cytogenetic or FISH analysis in 50% of chronic lymphocytic leukaemias (CLL). Trisomy 12 and del 13q14 account for 70% of these abnormalities. The incidence of these two abnormalities was studied in CLL patients by Southern blot analysis using a highly purified B-cell malignant population (CD5 > 95%, CD3 < 5%). Probes for the D13S25 marker on chromosome 13 band q14 and for the RBTN3 gene on chromosome 12 band p12-13, were used. Deletion of the D13S25 was detected in 17/42 patients (43%) in a homozygous (9.5%) or heterozygous (30%) configuration. Deletion of the D13S25 marker appears to be a clonal and early event in CLL development since it is detected in > 95% of the malignant clonal population. Conversely, trisomy 12 is rarely a clonal event (5/33 patients, 15%) and a varying proportion of cells carrying this abnormality can be demonstrated in 30% of CLL patients (10/33 patients).
- Published
- 1995
- Full Text
- View/download PDF
39. Defects in signal transduction pathways in chronic B lymphocytic leukemia cells.
- Author
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Jabbar SA, Hoffbrand AV, and Wickremasinghe RG
- Subjects
- Animals, Humans, Leukemia, Hairy Cell physiopathology, Leukemia, Lymphocytic, Chronic, B-Cell physiopathology, NF-kappa B physiology, Signal Transduction physiology, Transcription Factor AP-1 physiology
- Abstract
B chronic lymphocytic leukemia (B-CLL) and hairy cell leukemia (HCL) cells are refractory to many of the signals which activate normal B cells but are stimulated to proliferate by tumor necrosis factor (TNF). Cell signalling by TNF is mediated in part by the induction of the transcription factor families AP-1 and NF-kappa B. In some cellular contexts, these factors play a role in regulating cell cycle transit. AP-1 binds DNA as dimers of jun and fos family proteins and is regulated by a cascade of protein kinases which eventually activate a mitogen-activated protein kinase (MAP kinase) and also by protein kinase C. Three pathways have been implicated in the activation of NF-kappa B by extracellular ligands. 1, the activation of protein kinase C by diacylglycerol generated by ligand-mediated activation of phosphatidylcholine hydrolysis, 2, stimulation of specific protein kinases by ceramide generated following activation of a sphingomyelinase by diacylglycerol and 3, a novel pathway involving ligand-induced generation of free radical species. In B-CLL and HCL cells, the generation of nuclear-localized c-jun and c-fos proteins (components of AP-1) in response to TNF or PMA appears to be blocked. Whereas PMA failed to induce NF-kappa B in these cells, this factor was readily induced by TNF. TNF induction of NF-kappa B was abolished by antioxidants, suggesting involvement of the free radical pathway. The data discussed here suggest defects in coupling of some protein kinase C-dependent pathways in B-CLL and HCL cells and that TNF is able to bypass these blocks by the activation of NF-kappa B via a free radical-dependent pathway which is independent of protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1995
- Full Text
- View/download PDF
40. Inactivation of calcium ion-regulating inositol polyphosphate second messengers is impaired in subpopulations of human leukemia cells.
- Author
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Mengubas K, Jabbar SA, Nye KE, Wilkes S, Hoffbrand AV, and Wickremasinghe RG
- Subjects
- Adolescent, Adult, Aged, Aged, 80 and over, Bone Marrow enzymology, Child, Child, Preschool, Female, Hematopoietic Stem Cells enzymology, Humans, Immunoblotting, Inositol 1,4,5-Trisphosphate metabolism, Leukemia enzymology, Leukemia pathology, Leukocytes, Mononuclear drug effects, Leukocytes, Mononuclear enzymology, Lithium pharmacology, Male, Middle Aged, Precipitin Tests, Calcium metabolism, Inositol Phosphates metabolism, Leukemia metabolism, Phosphoric Monoester Hydrolases metabolism, Second Messenger Systems
- Abstract
Inositol 1,4,5-triphosphate (IP3) and inositol 1,3,4,5-tetrakisphosphate (IP4) are calcium-regulating second messenger molecules generated following the binding of a wide range of hormones and growth factors to their receptors. The actions of these messengers, which play important roles in the regulation of cell proliferation as well as in other signaling pathways, are terminated by the action of a 5-phosphomonoesterase (5-PME) enzyme. We have assayed this enzyme in normal and malignant hemopoietic cells. Extracts from normal bone marrow cells and peripheral blood mononuclear cells (PBMNC) degraded [3H]IP3 at rates of 74.5 (+/- 3.4) and 84.5 (+/- 7.9) pmol/min/micrograms protein, respectively. PME activity in 10/13 (77%) acute lymphoblastic leukemia samples were significantly below the normal range and the enzyme was completely undetectable in three (23%) of these. Enzyme activity in 8/9 (89%) chronic lymphocytic leukemia samples were below the normal range, being undetectable in three of these (33%). Nine of 24 (38%) acute myeloid leukemia samples contained low 5-PME levels, which was undetectable in one sample. Reduced 5-PME activity was detected in 2/7 (28%) of chronic granulocytic leukemia samples. The data here are consistent with the hypothesis that a reduced rate of degradation of IP3 and IP4 in some leukemia cells may result in the aberrant operation of signaling pathways, possibly including those involved in the control of cell proliferation.
- Published
- 1994
41. Redox reagents and staurosporine inhibit stimulation of the transcription regulator NF-kappa B following tumour necrosis factor treatment of chronic B-leukaemia cells.
- Author
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Jabbar SA, Hoffbrand AV, and Wickremasinghe RG
- Subjects
- Acetylcysteine pharmacology, Base Sequence, Butylated Hydroxytoluene pharmacology, DNA-Binding Proteins metabolism, Humans, In Vitro Techniques, Molecular Sequence Data, Oligodeoxyribonucleotides metabolism, Oxidation-Reduction, Staurosporine, Tetradecanoylphorbol Acetate pharmacology, Alkaloids pharmacology, Leukemia, B-Cell physiopathology, Leukemia, Hairy Cell physiopathology, NF-kappa B metabolism, Tumor Necrosis Factor-alpha pharmacology
- Abstract
B-chronic lymphocytic leukaemia (B-CLL) and hairy cell leukaemia cells (HCL) are refractory to stimulation by several cytokines which activate normal B-cells. However, tumour necrosis factor (TNF) promotes the proliferation of these cells. TNF regulates some of its cellular responses via the transcription factor NF-kappa B. Using an electrophoretic mobility shift assay, we demonstrate that TNF treatment of B-CLL and HCL cells in vitro resulted in the augmentation of NF-kappa B levels. In haemopoietic cell lines, TNF induction of NF-kappa B is mediated via the generation of reactive oxygen intermediates and by the activation of protein kinase C (PKC). We have used activators and inhibitors of these pathways to unravel TNF signalling in the cells of ten patients with B-CLL and two with HCL, using the increase in NF-kappa B levels following TNF treatment as an end point. Raising glutathione levels with N-acetyl cysteine substantially reduced NF-kappa B induction by TNF in two of four samples tested. These data suggest that redox mechanisms are involved in TNF signalling in these cells. Treatment with the PKC activator phorbol myristate acetate failed to activate NF-kappa B suggesting that this enzyme does not mediate the induction of NF-kappa B in these cells. However, the protein kinase inhibitor staurosporine inhibited TNF induction of NF-kappa B in four of five samples, suggesting that staurosporine-sensitive protein kinases (other than PKC) are involved in the signalling pathway. Our results suggest that PKC-independent pathways, including pathways sensitive to redox reagents, mediate the induction of NF-kappa B by TNF in chronic B-leukaemia cells. Additionally, these data suggest that defects in PKC-mediated pathways may contribute to the general reluctance of B-CLL and HCL cells to respond to mitogenic signals.
- Published
- 1994
- Full Text
- View/download PDF
42. Evidence that inositol phospholipids, but not choline phospholipids, are a potential source of growth-regulating second messenger molecules in HL60 leukaemia cells.
- Author
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Wickremasinghe RG, Porfiri E, and Hoffbrand AV
- Subjects
- Choline metabolism, Humans, Leukemia, Promyelocytic, Acute pathology, Phospholipase D metabolism, Tumor Cells, Cultured, Inositol metabolism, Leukemia, Promyelocytic, Acute metabolism, Phosphatidylcholines metabolism, Phospholipids metabolism, Second Messenger Systems physiology
- Abstract
Diacylglycerol is a potent second messenger which is generated via the cleavage of inositol- or choline-containing phospholipids and is involved in the transduction of proliferative signals. We have previously obtained evidence that the constitutive breakdown of inositol lipids may contribute to signalling the continuous proliferation of HL60 leukaemia cells (Porfiri E., Hoffbrand A. V. & Wickremasinghe R. G. (1991) Blood 78, 1069-1077). In order to assess the role of choline lipids as potential sources of growth-regulating second messengers, we have studied the pathways of constitutive breakdown of radiolabelled phosphatidylcholine in intact HL60 cells. Neither exponentially growing HL60 cells nor HL60 cells which had been induced to cease proliferation by treatment with dimethyl-sulphoxide degraded choline lipids via phospholipase C- or phospholipase D-catalysed pathways. Both pathways were, however, activated by phorbol myristate acetate irrespective of proliferation status. The data here suggest that, unlike inositol lipids, choline lipids are not a source of second messenger molecules with potential roles in the regulation of HL60 cell proliferation.
- Published
- 1993
- Full Text
- View/download PDF
43. MK 886, an antagonist of leukotriene generation, inhibits DNA synthesis in a subset of acute myeloid leukaemia cells.
- Author
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Khan MA, Hoffbrand AV, Mehta A, Wright F, Tahami F, and Wickremasinghe RG
- Subjects
- Adult, Aged, Child, Female, Humans, Leukemia, Myeloid, Acute pathology, Male, Masoprocol pharmacology, Middle Aged, DNA, Neoplasm biosynthesis, Indoles pharmacology, Leukemia, Myeloid, Acute metabolism, Leukotriene Antagonists
- Abstract
We have studied the actions of inhibitors of leukotriene generation on DNA synthesis (measured by 3H-thymidine incorporation) in blast cells from patients with acute myeloid leukaemia (AML). Cells from a subset only of these patients were sensitive to MK 886, a potent selective inhibitor of leukotriene synthesis. By contrast, DNA replication in cells from all of the patients was inhibited by nordihydroguiaretic acid (NDGA), a leukotriene synthesis inhibitor of lower selectivity. DNA synthesis in normal bone marrow cells and phytohaemagglutinin-stimulated lymphocytes was sensitive to NDGA but not to MK 886. The data suggest that NDGA inhibits DNA replication by a mechanism other than the abolition of leukotriene biosynthesis, but that DNA synthesis in a subset of AML cells may be dependent on the generation of lipoxygenase products, as indicated by sensitivity to MK 886.
- Published
- 1993
- Full Text
- View/download PDF
44. Antioxidants impair the coupling of cell-surface ligand receptors to the inositol lipid signalling pathway in human T lymphocytes but not in Jurkat T lymphoblastic leukemia cells. Evidence that leukotrienes are not involved in the coupling mechanism.
- Author
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Khan MA, Jeremy JY, Hallinan T, Tateson JE, Hoffbrand AV, and Wickremasinghe RG
- Subjects
- Arachidonic Acid metabolism, Cells, Cultured, Humans, Leukemia-Lymphoma, Adult T-Cell, Leukotrienes biosynthesis, Lymphocyte Activation physiology, Phosphoinositide Phospholipase C, Phosphoric Diester Hydrolases drug effects, Signal Transduction drug effects, Solubility, T-Lymphocytes metabolism, Tumor Cells, Cultured, Antigens, Differentiation, T-Lymphocyte drug effects, Antioxidants pharmacology, Leukotrienes physiology, Phosphatidylinositols metabolism, T-Lymphocytes drug effects
- Abstract
Ligands including phytohaemagglutinin (PHA) and anti-CD3 monoclonal antibodies trigger the generation of inositol lipid-derived second messengers following their binding to cell-surface structures of human T lymphoid cells. Previous evidence has suggested that the generation of leukotrienes may play an intermediary role in coupling the ligation of T lymphoid cell-surface structures to the inositol lipid signalling system in these cells (A.R. Mire-Sluis et al. (1989) FEBS Lett. 258, 84-88). Here we have studied the actions of two novel selective leukotriene biosynthesis inhibitors, MK 886 and BW A4C and of two general lipid soluble antioxidants, butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) on this pathway. Neither MK 886 nor BW A4C abrogated stimulation of inositol lipid breakdown following PHA or anti CD3 treatment of T lymphocytes. By contrast, this pathway was inhibited by BHT and BHA. These observations, together with our failure to demonstrate the generation of lipoxygenase products following PHA stimulation of T lymphocytes, suggests that an antioxidant-sensitive step other than the generation of leukotrienes plays a critical role in coupling cell-surface receptors to the inositol lipid signalling system in these cells. By contrast none of these inhibitors abrogated ligand-stimulated inositol lipid signalling in Jurkat T acute lymphoblastic leukaemia cells. These results suggest a heterogeneity in the organization of the signal transduction machinery in lymphoid cells at different stages of differentiation.
- Published
- 1993
- Full Text
- View/download PDF
45. Evidence that endogenous generation of leukotrienes does not regulate proliferation of malignant hemopoietic cell lines.
- Author
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Ayyub Khan M, Tateson JE, Hoffbrand AV, and Wickremasinghe RG
- Subjects
- 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine pharmacology, Blast Crisis, Burkitt Lymphoma drug therapy, Burkitt Lymphoma enzymology, Burkitt Lymphoma pathology, Cell Division drug effects, Cell Division physiology, Epoprostenol analogs & derivatives, Epoprostenol pharmacology, Hematopoietic System cytology, Hematopoietic System metabolism, Humans, Hydroxamic Acids pharmacology, Indoles pharmacology, Leukemia metabolism, Leukemia pathology, Leukemia, Myelogenous, Chronic, BCR-ABL Positive drug therapy, Leukemia, Myelogenous, Chronic, BCR-ABL Positive enzymology, Leukemia, Myelogenous, Chronic, BCR-ABL Positive pathology, Leukemia, Promyelocytic, Acute drug therapy, Leukemia, Promyelocytic, Acute enzymology, Leukemia, Promyelocytic, Acute pathology, Leukemia-Lymphoma, Adult T-Cell drug therapy, Leukemia-Lymphoma, Adult T-Cell enzymology, Leukemia-Lymphoma, Adult T-Cell pathology, Leukotriene Antagonists, Leukotrienes biosynthesis, Masoprocol pharmacology, Tumor Cells, Cultured, Benzeneacetamides, Hematopoietic System physiology, Leukemia drug therapy, Leukotrienes physiology, Lipoxygenase Inhibitors pharmacology
- Abstract
The proliferation of malignant hemopoietic cell lines is inhibited by antagonists of 5-lipoxygenase, suggesting that the endogenous generation of leukotrienes via the action of this enzyme may play some role in the proliferation of these cells (Snyder D. S., Castro R. & Desforges J. F. (1989), Expl Hemat. 17, 6). Here we have confirmed that the lipoxygenase inhibitors piriprost, nordihydroguiaretic acid and BW755C decreased DNA synthesis and proliferation of leukemic cell lines. However, the concentrations of these drugs required for half-maximal inhibition of proliferation were significantly greater than their IC50 values for 5-lipoxygenase inhibition. We therefore studied the actions of two novel, potent lipoxygenase inhibitors, BWA4C and MK886, on proliferation (as measured by estimating the number of viable, trypan blue-excluding cells) and DNA synthesis (measured by the incorporation of radiolabeled thymidine) in the leukemia cell lines HL60, K562 and Jurkat. Neither parameter was affected by concentrations of these drugs which were shown in parallel studies to substantially inhibit leukotriene generation in whole blood. The data show that endogenous leukotriene generation does not play a significant role in the regulation of proliferation of these leukemic cell lines and suggest that conclusions about leukotriene involvement in the control of cellular metabolic pathways based on the use of lipoxygenase inhibitors should be re-assessed.
- Published
- 1993
- Full Text
- View/download PDF
46. Do leukotrienes play a role in the regulation of proliferation of normal and leukemic hemopoietic cells?
- Author
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Wickremasinghe RG, Khan MA, and Hoffbrand AV
- Subjects
- Cell Division drug effects, Cell Division physiology, Hematopoietic Stem Cells drug effects, Humans, Leukotrienes pharmacology, Neoplastic Stem Cells drug effects, Neoplastic Stem Cells pathology, Hematopoietic Stem Cells cytology, Leukemia pathology, Leukotrienes physiology
- Published
- 1993
- Full Text
- View/download PDF
47. Impaired degradation of Ca(2+)-regulating second messengers in myeloid leukemia cells. Implications for the regulation of leukemia cell proliferation.
- Author
-
Nye KE, Riley GA, Poulter LW, Porfiri E, Hoffbrand AV, and Wickremasinghe RG
- Subjects
- Bone Marrow metabolism, Calcium metabolism, Cell Division physiology, Cell Fractionation, Humans, Leukemia, Myeloid pathology, Second Messenger Systems, Tritium, Tumor Cells, Cultured, Inositol 1,4,5-Trisphosphate metabolism, Leukemia, Myeloid metabolism
- Abstract
Inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate are Ca(2+)-regulating second messenger molecules which are generated via the cleavage of inositol lipids. We have previously shown that these species are autonomously generated in HL60 myeloid leukemia cells and that they may play a role in signalling the continuous proliferation of this cell line. Here we show that the activity of the 5-phosphomonoesterase (5-PME) enzyme which cleaves and inactivates these second messengers was strikingly reduced in HL60 cells compared to normal granulocytes or macrophages. Induction of differentiation of HL60 cells along the monocyte/macrophage or granulocytic pathways did not result in a significant increase in 5-PME activity. The activity of this enzyme was also low in extracts of bone marrow mononuclear cells from four patients with myeloid leukemia. A lesion in the 5-PME pathway may therefore result in the conservation of Ca(2+)-regulating second messengers in the HL60 cell line and in some myeloid leukemia cells. It is plausible that this lesion may co-operate with the autonomous cleavage of inositol lipids in the signalling of leukemic cell proliferation.
- Published
- 1992
48. Multiple tyrosine protein kinases structurally related to p56 lck are down-regulated following mitogenic stimulation of human T lymphocytes.
- Author
-
Hall BS, Hoffbrand AV, and Wickremasinghe RG
- Subjects
- Humans, In Vitro Techniques, Peptide Mapping, Signal Transduction, Down-Regulation, Isoenzymes metabolism, Lymphocyte Activation physiology, Protein-Tyrosine Kinases metabolism, T-Lymphocytes enzymology
- Abstract
p56 lck is a well-characterized tyrosine protein kinase (TPK) which is thought to play a role in mitogenic signal transduction in T lymphocytes. Immunoblot analysis of human lymphocyte proteins using an antiserum cross-reactive with phosphotyrosine resulted in the detection of a 55-60 kDa protein band (presumably p56 lck) as well as several additional phosphotyrosyl proteins in lymphocyte extracts. All of these phosphotyrosyl proteins were down-regulated following mitotic stimulation. Autophosphorylation of lymphocyte microsomal fractions in the presence of [gamma-32P] ATP resulted in the labelling of p56 lck as well as other proteins of different molecular weights. Analysis of these labelled proteins by tryptic digestion resulted in strikingly similar peptide maps. The data suggest that lymphocytes may contain a family of TPKs structurally related to p56 lck. The down-regulation of the putative TPKs following mitogenic stimulation of lymphocytes with phytohaemagglutinin suggests that this family of TPKs may participate in mitotic signalling events, followed by their down-regulation.
- Published
- 1990
- Full Text
- View/download PDF
49. Differential N-ethylmaleimide inhibition of two enzymes of the DNA alpha-polymerase-fraction from calf thymus.
- Author
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Wickremasinghe RG, Hesslewood IP, Holmes AM, and Johnston IR
- Subjects
- Animals, Cattle, DNA metabolism, DNA-Directed DNA Polymerase metabolism, Dose-Response Relationship, Drug, Kinetics, Magnesium pharmacology, Protein Binding, Thymus Gland enzymology, Ethylmaleimide pharmacology, Nucleic Acid Synthesis Inhibitors
- Published
- 1977
- Full Text
- View/download PDF
50. Inhibition by aphidicolin and dideoxythymidine triphosphate of a multienzyme complex of DNA synthesis from human cells.
- Author
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Wickremasinghe RG and Hoffbrand AV
- Subjects
- Aphidicolin, DNA-Directed DNA Polymerase metabolism, Dideoxynucleotides, Humans, Multienzyme Complexes metabolism, Thymine Nucleotides metabolism, DNA Replication drug effects, Diterpenes pharmacology, Thymine Nucleotides pharmacology
- Abstract
A multienzyme complex consisting of DNA polymerase and several DNA precursor-synthesizing enzymes was solubilized by gentle lysis of cultured human cells. This complex channelled the distal precursor [3H]dTMP into DNA. The patterns of inhibition of the complex by aphidicolin and dideoxythymidine triphosphate (ddTTP) suggested that the complex contained the replicative DNA polymerase, polymerase alpha. Inhibition by ddTTP was competitive with dTTP. This was exploited to estimate the effective concentration of [3H]dTTP at the site of DNA synthesis during channelling of [3H]dTMP into DNA. The estimated concentration (about 50 microM) was so high as to suggest that the solubilized complex was able to functionally compartmentalize DNA precursors.
- Published
- 1983
- Full Text
- View/download PDF
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